Overall goals of this research are two in number;(1) to continue the development of mass spectrometry instrumentation, software, and methodology for the identification and sequence analysis of proteins in complex mixtures at the attomole/femtomole level and (2) to employ the technology to solve structural problems at the cutting edge health related biological research. Specific Aims are 5 in number: (1) to develop instrumentation, software, and chemistry that will facilitate the use of electron transfer dissociation (ETD) mass spectrometry for the characterization, differential display, and quantitation of intact proteins (and their posttranslational modifications) expressed in two or more different cell trypes or states;(2) to decipher the histone code, patterns of post-translational modifications on histone variants that regulate transcription, gene and chromosome silencing, cellular differentiation and reprogramming, DMA damage/repair and chromosome condensation in apoptosis;(3) to immunopurify and characterize proteins that are posttranslationally modified by polyglutamylation;(4) to characterize novel antimicrobial, alpha- defensins/cryptdins and related cysteine-rich sequence (CRS) peptides by ETD mass spectrometry, and (5) to indentify components of kinetochore, the large multiprotein structure that coordinates chromosome segregation during cell division. Continued development of novel mass spectrometry technology is highly relevant to public health because it facilitates identification of proteins and their post-translational modifications that are uniquely expressed as a function of cellular perturbations such as disease, injury, drug treatment, hormone regulation, genetics and environmental impact. Enzymes responsible for introducing and removing post-translational modifications along with protein binding partners that recognize the post- translational modifications are implicated in many types diseases including cancer and autoimmune disorders and, thus, are targets for development of both disease diagnostics and candidates for therapeutic intervention.